Human reproductive health depends on gonad development, which can be adversely affected in utero by unknown genetic and environmental factors. In mammals, the Y-chromosome gene SRY initiates an incompletely understood network that directs the gonad to become a testis rather than an ovary. In nonmammalian vertebrates, other genes in the network determine sex, and in some species, environmental factors such as temperature or social cues can bias sex development. This project's broad objective is to learn how genetic factors, interacting with the environment, can tip the balance towards one sex or another. Work focuses on zebrafish, a genetically tractable vertebrate in which sex determination can be easily tipped in one direction or the other. The zebrafish gonad initially develops as a bipotential organ, with a few primordial germ cells in all juveniles initially developing as oocytes. In some individuals, oocytes die and the gonad becomes a testis; in others, oocytes survive and the gonad becomes an ovary. A key question is, what genetic or environmental signals cause presumptive oocytes to die in some individuals and to survive in others? An important tool to probe this question is a recessive mutation in fancl that causes female-to-male sex reversal. Preliminary experiments show excess cell death in homozygous mutant juvenile gonads and suggest the hypothesis that 1) environmental and genetic factors affect oocyte survival in juveniles, and thereby 2) alter the strength of an oocyte-derived signal that promotes, in surrounding somatic cells, 3) the production of aromatase, the enzyme that 4) converts testosterone to estrogen, which preserves oocytes and biases the gonad toward a female fate; 5) with insufficient quantities of the hypothesized oocyte-derived signal, the gonad becomes a testis. Aim 1 is to identify genetic factors linked to sex phenotype in F2 mapping crosses. Aim 2 is to conduct a genome-wide search for genes expressed sex specifically by comparing transcriptomes of all-male populations of zebrafish and medaka, a species with genetic sex determination, to female-containing sibling populations (Aim 2a) and to investigate expression patterns of candidate sex determination genes, steroid receptor genes, and extracell signaler genes (Aim 2b). Aim 3 is to test additional components of the hypothesis by learning whether blocking the retinoic acid signal for entry into meiosis can cause sex reversal (Aim 3a) and whether primordial germ cell number controls sex development in zebrafish and medaka (Aim 3b). The proposed work has significance for it's potential to identify new genes and new gene functions by comparing a vertebrate with a delicate balance for sex differentiation (zebrafish) to one with a known genetic male determinant (medaka). Results will contribute to a better understanding of the molecular genetic nature of the vertebrate sex-balance mechanism, and thus contribute to our understanding of possible mechanisms for the current increase in human reproductive disease, including testicular dysgenesis syndrome and polycystic ovary syndrome increasingly observed in developed countries. PUBLIC HEALTH RELEVANCE: Understanding the biological mechanisms that tip the balance of sex determination between male and female is essential to understand recent increases in human reproductive disorders originating in the womb, including sterility and testis cancer in men and polycystic ovary syndrome in women. Certain genetic make- ups may be especially prone to disruption. Investigations of vertebrate species with finely balanced sex determination mechanisms may provide insight into how environmental and genetic factors tip the sex determination balance in humans. Although many features of zebrafish gonad development are similar to those in humans, zebrafish sex determination is more labile; furthermore, in zebrafish, genetic tools are available to dissect the mechanisms of sex determination, which should identify new genes and new gene functions relevant for human reproductive health.